Post-transcriptional regulation of messenger RNA (mRNA) stability and translation is an important mechanism for rapidly controlling gene expression in response to stimuli, including environmental changes. This project seeks to generate and utilize structural information to enhance our understanding of these processes with an emphasis on the importance of RNA target specificity for proper gene regulation. Typical PUF proteins are sequence-specific RNA-binding proteins that are important regulators of gene expression for embryonic development and germline stem cell maintenance. Beginning with determining the first crystal structure of a PUF protein in complex with RNA to recent work on the specificity of human, yeast, and C. elegans proteins, we have identified both common and unique features of RNA recognition by this family of proteins. The combination of the features in any particular protein results in a unique network of mRNAs that are regulated by that protein. In this fiscal year, we have advanced this work by identifying and studying the plasticity of RNA recognition by PUF proteins. We have identified a feature called an upstream cytosine binding pocket that can refine RNA target specificity of particular PUF proteins. We now identify structural features that allow for more diversity of RNA recognition by a single PUF protein. We have determined crystal structures of a single PUF protein with multiple RNA recognition sites. These structures reveal how this protein can accommodate diverse recognition sequences on a common framework. Thus PUF proteins can utilize both narrowed and broadened specificity to appropriately regulate genome expression. We are also studying the role of phosphorylation to regulate the affinity of a stem-loop RNA binding protein. We have used a combination of structural, biochemical, computational and biophysical methods to demonstrate that phosphorylation can stabilize an intrinsically disordered protein to promote RNA binding and can reduce the entropic barrier to binding.